Project Summary/Abstract Of the average 57 million annual deaths over the past ten years, about 14.8 million are directly due to infectious diseases with millions more due to secondary effects of infections (64). The numbers of annual deaths due to infections by Salmonella Typhi and S. Paratyphi A, hepatitis B virus (HBV), Streptococcus pneumoniae and Mycobacterium tuberculosis (Mtb) vary widely but may account for about 40 percent of the total deaths with an even greater cost being due to the severe morbidity associated with these diseases (241). In the belief that improving health, nutrition and economic well being (the latter dependent on the first two) provides the best means to enhance the quality of life globally and thus reduces conditions that result in warlike and terrorist behavior, we propose a vaccine development program based on our recent technical developments in using recombinant attenuated Salmonella vaccines (RASV). Our Specific Aims are (i) to enhance induction of mucosal and systemic antibody responses to RASV-expressed protective pneumococcal antigens (as a model antigen) by increased production and release of membrane vesicles and by altering RASV cell adherence attributes, (ii) to enhance biological containment of live RASV strains by ensuring inability to persist and/or to completely lyse in vivo and especially in the intestinal tracts of birds and animals, (iii) to design and construct S. Paratyphi A as an antigen and DNA vaccine delivery vector with a diversity of genetically-specified features to ensure safety and efficacy when used to immunize infants and adults to protect against enteric fever and an additional infectious disease such as caused by HBV, (iv) to design, construct and evaluate a recombinant attenuated S. Paratyphi A vaccine exhibiting regulated delayed lysis within effector lymphoid tissues in the immunized individual to deliver hepatitis B virus (HBV) core particles with fused preS1, preS2 and T-cell epitopes and a DNA vaccine encoding small S antigen for synthesis in cells of the immunized host with features to enable delivery soon after birth, and (v) to design, construct and evaluate a recombinant attenuated S. Typhi vaccine to prevent infections by Mtb using delivery of protective antigens by Type II and III secretion and/or by regulated delayed lysis in vivo with lysis occurring in cell compartments to enhance antigen presentation either by MHC Class I or Class II and by regulated delayed lysis in vivo in the cytoplasmic compartment to release a DNA vaccine optimized for nuclear targeting leading to synthesis and modification of protective antigens by the immunized individual. We will also develop our Master File, prepare and fully characterize candidate vaccine Master Seeds for stability and safety, prepare and submit protocols for IRB approvals, submit information necessary to obtain INDs, and perform any other work needed to arrange that the best candidate vaccines be clinically evaluated in human volunteers. Project Narrative The research proposed will further develop and refine technologies for design and construction of live recombinant attenuated Salmonella vaccine (RASV) vectors to deliver after needle-free oral inoculation protective antigens and/or DNA vaccines to stimulate mucosal, systemic and cellular immunities against bacterial, viral and parasite infectious disease agents. More specifically, we will develop a safe, efficacious inexpensive vaccine using S. Paratyphi A to deliver multiple hepatitis B virus (HBV) antigens for oral immunization of newborns and infants to prevent HBV transmission during childbirth. We will also develop a RASV using S. Typhi as vector to prevent transmission and infection by Mycobacterium tuberculosis.